Composition containing useful substances originating in fishes and shellfishes and process for the preparation of the substances

ABSTRACT

A process for producing water-insoluble substances derived from fishes and shellfishes, includes the treating of the waste of fishes and shellfishes containing the substances with proteolytic enzymes under stirring to obtain an oil-in-water (O/W) type emulsified composition. This composition contains water-soluble amino-acids, oligoproteins having a molecular weight of not greater than 30,000, water-soluble minerals, water-insoluble highly unsaturated fatty acids and proteins (solid matter) having a molecular weight of 20,000 to 100,000, with 50% or more of all the proteins in said emulsified composition having a molecular weight of 20,000 to 100,000. Thereafter the emulsified composition is separated into solid and liquid phases, and the obtained solid composition containing proteins with a molecular weight of 20,000 to 100,000 and fats and oils is extracted with an organic solvent.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP98/04789 which has an Internationalfiling date of Oct. 22, 1998, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a process for separating thewater-insoluble (oil-soluble) useful substances economically from thewastes of fishes and shellfishes. More particularly, it relates to thesaid process featuring the steps of preparing a novel oil-in-water typeemulsified composition from the wastes of fishes and shellfishes bytreating the wastes with proteolytic enzymes to convert proteins in thewastes into oligoproteins having a molecular weight in a specifiedrange, separating said composition into solid and liquid, and extractingthe solid matter with an organic solvent.

BACKGROUND ART

It is needless to say that fishes and shellfishes are natural resourceswhich are of vital importance as a nutrient for mankind. These fishesand shellfishes are processed in a variety of forms, and in the courseof processing, their skins, guts, bones, etc., are discarded asindustrial waste. Although part of such waste is utilized as fertilizeror stock feed, most of it, under the present situation, is leftunrecycled and necessitates disposal at a great cost. The regulations onthe method of disposal have become more rigorous recently, and thetreatment of such waste is posing a serious problem from the aspect ofenvironmental protection.

For instance, cuttlefish skin, which is discarded as industrial wasteafter hot-water processing, is estimated to produce at least 2,000 tonsof waste per year in Japan. Recycling of such cuttlefish skin waste willbe a great benefit to the prevention of environmental pollution in thesense of effective utilization of industrial waste. Also, by promotingrecycling of waste matter which is presently disposed at a greatexpense, it will become possible to recover useful substances at anotably low cost.

It is known that these fish and shellfish wastes (skin, guts, bone,head, tail, fin, eyes of scallops, shell, and sometimes whole fish orshellfish body) contain various useful substances in large quantities orin low or only slight concentrations.

For instance, as water-soluble substances, various types ofwater-s,oluble vitamins, various types of minerals, polysaccharides,amino-acids, chondroitin sulfuric acid, enzymes, protamine,triglycerides of saturated or unsaturated lower fatty acids, etc., areknown to exist in said wastes, and as water-insoluble substances,calcium, triglycerides of saturated higher fatty acids, triglycerides ofunsaturated fatty acids (monoenoic acids such as myristoleic acid,palmitoleic acid and nervonic acid, tetraenoic acids such as arachidonicacid, pentaenoic acids such as clupanodonic acid, hexaenoic acids suchas docosahexaenoic acid, etc.), phospholipids such as phosphaditidylcholine and phsphaditidyl ethanolamine, etc., are known to be containedin said waste.

These substances can be applied to a wide scope of uses, such asnutrition promoting foods, humectants, liquid crystal material,medicinal base material, antibacterial agents, preservatives, stockfeed, etc., according to the particular properties of the substances.

Extraction from the natural products may be the best way for obtainingan desired compound with ease and at low cost if the selection of rawmaterial is appropriate and an effective means for extraction iselaborated.

For example, an extensive search has been made for the materialscontaining docosahexaenoic acid (hereinafter abbreviated as DHA) in highconcentrations, and the methods for preparing DHA from the head, guts orwhole of the blue-back fishes such as tuna, bonito, saurel, macherel,sardine, etc., have been proposed (JP-A-63-164852 and JP-A-64-50890).

However, in view of the difficulty of the separating operations,attempts for yielding the objective useful substances are also beingmade using the artificial or natural materials other than fishes andshellfishes by introducing such techniques as synthetic reactions orenzymatic reactions for certain types of substances.

For instance, it has been reported that the glyceride esters of highlyunsaturated fatty acids were synthesized by acyl group exchange betweenthe fatty acids with a lipase (Journal of Fermentation andBioengineering, Vol. 69, pp. 23-25, 1990).

Also, the techniques for obtaining a desired phospholipid derivative byenzymatic ester exchange or acylation from phospholipid orlysophospholipid and an optional fatty acid or fatty acid ester havebeen disclosed (JP-A-63-105686, JP-A-63-185391, JP-A-2-35093 andJP-A-5-236974). However, all of the above-mentioned reports or patentsinvolve many problems, such as random entry of the introduced fatty acidinto the sn-1 or sn-2 position and too low enzyme reaction rate, forrealizing practical industrial application. Thus, a technique enablingextraction of a desired useful substance from selected waste containingsuch a substance in a higher concentration by a simple process has beendesired.

A method featuring extraction of the desired substance from amicroorganism cultured in a DHA-added medium has been also disclosed(JP-A-5-123176). Further, a method of preparing DHA from the guts ofcuttlefish (JP-A-2-8298) and a method of preparing DHA from cuttlefishskin (JP-A-6-321970 and JP-A-9-77782), worked up in view of high DHAconcentration in phospholipid of cuttlefish, have been reported.

In order to separate the useful substances from the fish body, generallythe fish body is first divided into the water-soluble components and thewater-insoluble components.

Usually steaming is employed for this treatment. When steam is passedthrough waste of fishes or shellfishes, oil is separated out. In thiscase, however, the interface between water and oil is ambiguous and itis hard to distinguish between the two phases, so that no perfectseparation can be achieved and, in some cases, the whole sets to gel,making it impossible to carry out the separating operation.

Also, according to the above method, since steam heating is applied, theuseful components which are unstable to heat may be denatured and loosetheir activity. Generally, the fish body components are prone to oxidizeby the action of oxygen in the air to generate the so-calledfish-smelling components (amines, aldehydes, etc.). Needless to say, thepresence of such fish-smelling components greatly reduces the commercialvalue of the product. Lots of labor and cost (for high-degree vacuumdistillation, etc.) are required for removing the fish smell. Thesimilar problems are also raised in connection with the tinted matterwith heating.

In order to remove water which accounts for about 90% of the overallfish body weight, it is practiced to dry the fish body by variousmethods such as lyophilization, spray drying, sun-drying, etc., andextract the useful substance from the dried product with a solvent(hexane, alcohol, etc.). This method, however, requires specificequipment such as a dryer, which leads to high production cost. And whenheating is needed, there arises the problem of the denaturing of theuseful components as mentioned above. Further, there may be situationswhere it is required to transport the material-to-be-processed with highwater content to a treating plant which may not necessarily be locatedadjacent to the waste generating plant. Moreover, in case a refrigeratorvan or such is used for the transport, it is necessary to takesufficient measure to prevent putrefaction (if putrefaction begins, thecommercial value of the material will be spoiled by the fish smell asmentioned above), and also the transportation cost will become a burden.Therefore, a drying treatment for removing water is usually conductedbefore extraction of the desired substance.

As means for removing water from a living specimen, various methods suchas compression, air drying, heat drying, vacuum drying and freeze-dryingare conceivable. For reducing water without changing the normal figureof the living specimen, compression alone is insufficient to attain adesired water removal rate while air drying, heat drying and vacuumdrying involve the problem in respects of treating temperature andtreating time. The operation cost of the freeze-dryer required for thesaid water removal is vast. In order to elevate the drying efficiency,the surface area of the specimen needs to be enlarged as much aspossible. There is also required extra labor for feeding of the materialto the freeze-dryer. Further, a freeze-dryer takes a long time tooperate. The equipment investment for the freeze-dryer tends to be tooheavy for elevating the production capacity. For these reasons,employment of the freeze-drying step in an industrial process isdisadvantageous. Thus, a simple and inexpensive dehydration method thatcan replace the conventional freeze-drying treatment without affectingthe product quality has been desired.

Certain types of waste of fishes and shellfishes, for instancecuttlefish skin, are covered on their surface with a slimy substancecomposed of a polysaccharide. This substance is often responsible for alarge reduction of the filtering rate in the filtration step that isconducted in the process of phospholipid extraction with a solvent.

The present invention provides the techniques for easily andeconomically extracting the water-soluble and water-insoluble usefulsubstances from the waste of fishes and shellfishes under the mildoperating conditions that will nct cause denaturing of these usefulsubstances.

We have made studies in search of the solution to the various problemsmentioned above. We have treated waste of fishes and shellfishes byusing various types of proteolytic enzymes to degrade proteins,centrifuged them to separate the paste and the extract according to thedegree of proteolysis, and investigated the condition of separation ofoils and fats.

As a result, it was found that when an emulsified material obtainedafter treating waste with a certain combination of proteolytic enzymeswas centrifuged, the material could be definitely separated into thephase of aqueous solution substantially free of oils and the sedimentportion containing oils.

More specifically, when waste of fishes or shellfishes was treated byusing specific proteolytic enzymes to degrade proteins in the waste tomake a mixture of the water-soluble low-molecular weight peptides andamino-acids and the water-insoluble high-molecular weight peptides, themixture was formed in a state of emulsion that has never been reportedbefore. When this novel emulsified material was centrifuged by anordinary method, it was easily separated into the sediment containingoils and the aqueous layer substantially free of oils. In the sedimentobtained in the manner described above, there were containedwater-insoluble proteins and fats, and about 90% of the whole fats andoils was recovered as sediment. It was confirmed that the collected fatsand oils contained triglycerides and phosphaditydiyl choline of thehigher unsaturated fatty acids such as eicosapentanic acid anddocosahexaenoic acid. The above finding underlies the present invention.

DISCLOSURE OF THE INVENTION

The present invention is directed to the following embodiments:

(1) A process for producing water-insoluble useful substances derivedfrom fishes and shellfishes, which comprises the steps of:

(a) treating waste of fishes and shelifishes containing usefulsubstances with a proteolytic enzyme(s) under stirring to obtain anoil-in-water (O/W) type emulsified composition comprising (i)water-soluble components which comprise water-soluble amino-acids,oligoproteins having a molecular weight of not greater than 30,000,vitamins and water-soluble minerals such as salts, and (ii)water-insoluble components, as solid matter, comprising oils and fatscontaining water-insoluble highly unsaturated fatty acids and proteinshaving a molecular weight of 20,000 to 100,000; wherein 50% or more ofall the proteins in the emulsified composition have a molecular weightof 20,000 to 100,000;

(b) separating said emulsified composition into solid and liquid phasesto obtain a solid composition; and

(c) extracting said solid composition with an organic solvent.

(2) An oil-in-water (O/W) type emulsified composition obtained bytreating waste of fishes and shellfishes containing useful substanceswith a proteolytic enzyme(s) under stirring, which comprises (i)water-soluble components comprising water-soluble amino acids,oligoproteins having a molecular weight of not greater than 30,000,vitamins and water-soluble minerals such as salts, and (ii)water-insoluble components, as solid matter, comprising oils and fatscontaining water-soluble highly unsaturated fatty acids and proteinshaving a molecular weight of 20,000 to 100,000, wherein 50% or more ofall the proteins in the emulsified composition have a molecular weightof 20,000 to 100,000.

(3) A novel solid composition comprising proteins having a molecularweight of 20,000 to 100,000 and oil and fat matter, obtained byseparating the emulsified composition set out in (2) above into solidand liquid phases.

(4) A process as described in (1) above, wherein at least two enzymesselected from 1) an endo-type proteolytic enzyme, 2) an exo-typeproteolytic enzyme and 3) an endo- and exo-type proteolytic enzyme areused in combination as said proteolytic enzymes.

(5) A process as described in (1) above, wherein the molecular weight ofthe proteins after the treatment is 30,000 to 50,000.

(6) A solid composition as set out in (3) above, wherein 50% or more ofthe whole protein after the treatment has a molecular weight of 30,000to 50,000.

(7) A process as described in (1) above, wherein the waste is cuttlefishskin.

(8) A process as substantially described in (1) above, wherein the wasteis eyeballs or spawn of salmon or tuna.

(9) A process as described in (1) above, wherein the useful substance isa phospholipid type highly unsaturated fatty acid.

(10) A process as described in (1) above, wherein the useful substanceis phospholipid type DHA.

(11) A process as described in (1) above, wherein the solid matter afterseparation into solid and liquid phases is dried and then extracted withan organic solvent.

(12) A process as described in (1) above, wherein the organic solvent isselected from the group consisting of ethanol, hexane, acetone and amixture thereof.

(13) A water-insoluble useful substance derived from fishes andshellfishes, obtained by extracting the solid composition described in(3) above with an organic solvent.

(14) An edible composition containing 35% or more of a phospholipid(s),a highly unsaturated fatty acid(s) in an amount of 40% or more based onthe whole fatty acids in the phospholipid(s), and proteolyticenzyme-treated matter of fishes and shellfishes.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, as the first step, waste of fishes orshellfishes containing the objective useful substances is treated with aproteolytic enzyme(s) under stirring. In this case, it will be expedientto crush the waste depending on its form, state of existence, etc., assuch crushing increases the surface area of the waste matter to betreated to allow more smooth proceeding of the enzyme reaction. Powderywaste may not require such pre-treatment. In the present invention,crushing and stirring can be well effected by the commonly used means,and no specific devices are required therefor.

Generally, this type of waste can be easily crushed as it is usuallykept frozen in order to prevent putrefaction. Crushing and stirring areperformed for the purpose of promoting smooth progress of the enzymereaction, and to this end it suffices to crush the material (waste) to agrain size of about 0.01 to 0.1 cm. For instance, a commercial foodprocessor available from Nakabe Corporation can be conveniently used forthis purpose. Some types of waste contain bone tissues such as brokenpieces of bone and shells, but since they settle down as a sedimenttogether with oils and fats, they won't disturb the process of thepresent invention. In the case of the waste with a low water content,such as the eyeballs of tuna, it is advisable to add water for elevatingfluidity so as to increase the efficiency of contact with enzymes. Thewater added is separated from the sediment, so that it will not obstructextraction of the useful oil and fat material, allowing the fulfillmentof the object of the invention.

As the stirring means, it is possible to use an ordinary stirrerprovided with one or two turbine or propeller type stirring vanes. Thestirrer capacity, although variable depending on the shape of the blade,the size of the reactor used and the nature of the liquid to be treated,is preferably around 20 to 100 rpm when using a reactor with a volume ofabout 1 m³. Technologically, the power required per unit volume of thematerial to be stirred is referred to as a practical measure of stirringefficiency. For the reaction in the present invention, such stirringefficiency preferably falls in the range of about 0.5 to 5 KW/m³.Stirring too slowly may cause accumulation of the crushed waste sedimentin the reactor which retards the progress of the reaction. Stirring toostrongly may cause the formation of a creamy emulsion which greatlyhinders solid-liquid separation in the next step.

The proteolytic enzymes which can be used in the present inventioninclude endo-type peptidases (enzymes of the type which breaks theprotein linkage at its middle point), exo-type peptidases (enzymes ofthe type which breaks the protein linkage at an end; the enzyme whichbreaks the linkage at its N end is called aminopeptidase, and the enzymewhich breaks the linkage at its C end is called carboxypeptidase), andendo- and exo-type peptidases having the functions of both endo-type andexo-type enzymes. In the present invention, it is more effective to usea mixture of two or more types of said enzymes than to use only onetype.

The concentration of the proteolytic enzymes used in the presentinvention is 0.1 to 10% by weight, preferably 0.5 to 2% by weight basedon the overall amount of protein. The enzyme reaction conditions shouldbe selected with care according to the type of the waste to be treatedfor obtaining a desired form of emulsion. It is to be noted that if theproteolytic reaction of protein proceeds to excess to reduce the proteinmolecular weight to less than 20,000, the aqueous phase/oil phaseseparation becomes difficult to attain, making it impossible to obtainan emulsified composition having the effect of the present invention.Also, if the post-reaction residual protein becomes small in quantity ascompared to the oils and fats, the latter may not be sufficiently takenup in the solid, or oil may ooze out during drying of the obtained solidsediment, giving rise to problems, such as cumbersome handling of thematerial. Of course, it is impossible to obtain the effect of thepresent invention when degradation has advanced perfectly to theformation of an amino acid or an oligo-protein akin thereto.

Conversely, if proteolysis is insufficient and the molecular weight ofprotein stays above 100,000, it is also impossible to derive the effectof the present invention.

Gel filtration, SDS electrophoresis and other methods are known for thedetermination of molecular weight. Gel filtration is simple andpractical, but one needs to pay attention to the interaction with thecarrier. When using electrophoresis, the molecular weight can bedetermined by using a mixture of phospholipase b (MW 94,000), albumin(MW 67,000), ovalbumin (MW 43,000), carbonic anhydrase (MW 30,000),trypsin inhibitor (MW 20,000) and α-lactalbumin (14,000) as an MWmarker. It is also possible to determine the amount of protein, alongwith its molecular weight, from the degree of color development ofprotein in the electrophoresis. The average molecular weight of proteinin fishes and shellfishes is usually around 600,000, but in order toobtain the effect of the present invention, it is imperative to make acomposition in which 50% or more, preferably 70% or more of the wholeprotein has a molecular weight in the range of 20,000 to 100,000, morepreferably 30,000 to 50,000, by the techniques of the present invention.

Oligopeptides having a molecular weight falling in the above-definedrange have an emulsion-forming effect, that is, its water-solublecomponent and the oil-adsorbed solid protein cooperate to form anemulsified state. The present inventors found that in this operation,quite surprisingly, oil is scarcely distributed to the aqueous layer andforms a mixture with the solid protein. Consequently, the aqueous layerand the oil layer can be separated distinctly from each other by asimple solid/liquid separating method. Since oil settles down withsedimentation of solid protein, it can be easily separated from theaqueous layer.

Solid/liquid separation can be effected by using a spontaneouslysettling thickener or a filter, but it is preferred to use a centrifugewhich allows faster treatment. The basket type and decanter typecentrifuges can be used. Industrially the decanter type is preferred asit is capable of continuous treatment.

The enzyme reaction conditions for degrading protein into theabove-defined range of molecular weight needs to be set in considerationof the optimal temperature or heat resistance of the enzymes used andthe type of the waste to be treated as mentioned before, but usually thereaction temperature is 30 to 80° C. and the reaction time is 0.5 to 5hours, preferably the reaction temperature is 30 to 50° C. and thereaction time is 1 to 2 hours. An antioxidant such as vitamin C,tocopherol, vitamin E cr catechin may be added to the reaction system tosuppress oxidation during the reaction. In order to prevent the enzymereaction from advancing more than necessary, the enzymes are preferablydeactivated after the desired emulsified state has been obtained. Thiscan be usually effected by heating at 80 to 100° C. for 5 to 20 minutes.

In the thus obtained emulsion, the water-insoluble proteins are degradedby the enzymes while the water-soluble amino-acids and low-molecularweight peptides are increased. Also, the saccharides in the cells andthe salts which have been combined with proteins are made soluble inwater. The normally water-soluble components are transferred to theaqueous layer while the water-insoluble ones are shifted to the sedimentlayer. Said water-insoluble oils and fats have substantially beenrecovered in the sediment.

The water content in waste of fishes and shellfishes is usually 80 to90%, but the sediment obtained in the manner described above is about 2to 10% of the pre-treatment waste in weight, and the water content ofsuch a sediment is about 40 to 70%, usually around 65%. Thus, the watercontent per unit amount of the useful substances is greatly reduced incomparison with the pre-treatment waste, which signifies a remarkablereduction of cost and labor required for drying removal of water inseparation and extraction of oils and proteins.

The wet sediment is preferably dried to a water content of 10% or less,preferably 2% or less, because of elevated extraction efficiency. Inthis case, too, the cost for drying is drastically lessened as theuseful substances are concentrated in the sediment and reduced inquantity.

Because of the enzyme reaction, no severe heating such as that conductedin steaming is necessary, and the desired concentration can be effectedwithout causing denaturing of the useful substances which are unstableto heat.

The sediment is extracted with an organic solvent (chloroform,dichloromethane, methanol, ethanol, acetone, hexane, toluene, etc.). Ifnecessary, it may be dried before extraction. An effective solvent orcomposite solvent is selected in consideration of the type of the wasteto be treated, the type and amount of the objective useful substance(s)and other factors. For instance, ethanol, hexane and the like areeffective for extracting DHA phospholipids. In the extract obtained bythe process of the present invention, phospholipids are contained at ahigh rate of over 35%, and the higher unsaturated fatty acids such asdocohexaenoic acid and eicosapentaenoic acid are also contained at arate of over 40% of the whole fatty acids. It is also a merit to thisinvention that high-purity phospholipids can be extracted even from thewaste material which may not necessarily be high in phospholipidconcentration. Further, the lipids obtained according to the presentinvention contain small quantities of oligoproteins produced by theproteolytic enzymes and various kinds of phospholipids, so that thecomposition of the present invention is expected to possess excellentphysiological activity when used in foods. It is known that some of thesubstances obtained from enzymatic treatment of fish proteins have agentle blood pressure reducing action. Such physiologically activesubstances often exhibit a physiologically more desirable effect whenmixed with other materials than when they are present singly with highpurity. Such an effect is often seen in various kinds of herbs.Therefore, when using the useful substances of the present invention asa component of health foods, a more desirable effect may be expectedfrom a purity of such a standard as obtained by extraction. Theextracted active substances may be further purified by the conventionalmethods such as recrystallization, chromatography, filtration, etc.,which are properly selected according to the situation, to obtain ahigh-purity physiologically active substance which can be offered, forinstance, to medicinal and pharmaceutical uses. The phospholipidsmentioned above can also be enhanced in purity by crystallizing theirhexane extract with acetone which is a poor solvent of phospholipids.

The sediment is dried and then, if necessary, ground to give a powderrich with useful substances such as DHA. Therefore, the powder can beused either as a material from which to extract the useful substanceswith higher purity or may be used as it is as functional foods or feedfor aquarium fishes, cattle, pets, etc. Since the product contains theuseful substance(s) and is of powdery form, it is easy to handle andunsusceptible to air oxidation and other forms of denaturalization. Theseparated aqueous phase portion also contains many useful substances, sothat it can be used in the form as it is (extract from fishes andshellfishes) as foods or feed for aquarium fishes, pets, etc. Thisextract can also be used as a material from which to further extract theuseful substance(s) by suitable means such as solvent extraction orchromatography to obtain a high purity product. As described above, thepresent invention provides the techniques for economically separatingthe water-soluble or water-insoluble useful substances contained in thewastes of fishes and shellfishes.

The present invention will be further illustrated by the followingexamples and comparative examples, but the invention is not subject toany restrictions by these examples.

EXAMPLE 1

The frozen cuttlefish skin was crushed by a food processor (mfd. byNakabe Corp.) and 1,000 g thereof was weighed into a 2,000 ml beaker.The beaker was fixed in a water bath, and a variable-speed stirrer (mfd.by EYELA Inc.) provided with a stirring rod was set in the center of thebeaker.

The crushed mass of cuttlefish skin was heated to 50C with stirring at500 rpm. When the mass being stirred reached 50° C., there were addedthereto 0.3% of endo-type Alkalase (produced by Novo Industry Ltd.),0.3% of exo-type Amano M (produced by Amano Pharmaceutical Co., Ltd.),0.3% of exo- and endo-type Amano A (produced by Amano PharmaceuticalCo., Ltd.) and 0.1% of deaminase (produced by Daiwa Chemical Co., Ltd.)for improving the taste and smell of the water-soluble substanceexpected to be yielded, viz. the extract, each of said additives beingdissolved in a small quantity of water (% being based on the material tobe treated).

Vitamin E was also added in an amount of 0.3% based on the oils and fatscontained in the mass. Then the mass was subjected to an enzyme reactionfor 2 hours maintaining the temperature at 50° C., followed byadditional 15-minute treatment at 90° C. to deactivate the enzymes.

After the enzyme reaction, the resulting solution (O/W type emulsifiedcomposition) was diluted and subjected to electrophoretic assay, whichshowed approximately 75% of the original proteins had been made topossess a molecular weight of 31,000 by the above treatment.

The thus obtained emulsified composition was cooled, put into a tube ofa high-speed centrifuge (mfd. by Hitachi Ltd.) and centrifuged at 10,000rpm for 20 minutes to separate the composition into solids andwater-soluble matter.

On conclusion of centrifuging, the centrifuge tube was taken out, thewater-soluble matter was transferred to a beaker, and 120 g of thesediment was scraped out, transferred to a stainless steel-made platevat and, with the sediment deposited to a thickness of 3 to 5 mm on thevat, placed in a 50 to 60° C. dryer (mfd. by Tabai Co., Ltd.) where itwas dried for 12 hours. The dried product was removed from the vat andapplied to a small-sized grinder (mfd. by Iwatani Co., Ltd.) to give 31g of powder.

As a result of the above operations, the following data were obtainedfrom the separated solids and water-soluble matter. Water content wasdetermined by the Karl-Fischer method and oil content was determined bythe conventional gravimetric method involving chloroform extraction. Thedata obtained from similar determinations of raw cuttlefish skin anddried powder thereof are also shown.

Water Oil Raw cuttlefish skin 92.2% 1.5% Dry powder of cuttlefish skin1.2 19.8 Dry powder of solid composition 0.8 32.9 of the presentinvention Water-soluble matter 93.5 0.2 or less

As is seen from the above table, the oil and fat content in the drypowder of the solid composition of the present invention is higher by21.9 times and 1.7 times, respectively, than those in the raw cuttlefishskin and in the powder obtained by simply directly drying the cuttlefishskin. It will be also seen that oils and fats are scantly in thewater-soluble matter. A quantitative analysis of oil in the dry powderby YEATROSCAN confirmed the presence of 12.7% of phosphaditidyl cholineand 8.3% of phosphaditidyl ethanolamine. Gas chromatographicdetermination of fatty acids after hydrolytic ester exchange showed thepresence of 38.1% of docosahexaenoic acid and 10% of eicosapentaenoicacid. This corroborates that docosahexaenoic acid of phosphaditidylcholine and eicosapentaenoic acid derivatives had been extracted in highconcentrations in the solid composition centrifuged according to themethod. of the present invention.

In a similar analysis of 9 g of a cake obtained by subjecting the drypowder of said solid composition to ethanol extraction and, afterdrying, hexane extraction, followed by crystallization and sedimentationwith acetone which is a poor solvent of phosphaditidyl choline, andfinally filtration by Nutsche funnel, it was confirmed that the cakecontained 38.5% of phosphaditidyl choline and 11.4% of phosphaditidylethanolamine. Further, the result of a fatty acid analysis showed thepresence of 39.5% of docosahexaenoic acid and 13% of eicosapentaenoicacid. It was thus possible to obtain high-purity phosphaditidyl cholineand phosphaditidyl ethanolamine.

Comparative Example 1

The same procedure as in Example 1 was carried out except that thereaction time was prolonged to 4 hours. As a result, the amount of thesolids obtained after centrifuging was as very small as 5 g. Also, theoil content in the obtained solids was 8%, and it was impossible torecover a sufficient amount of oil. Electrophoretic analysis of theenzyme reaction solution showed 87% of the original proteins had amolecular weight of not greater than 15,000.

Comparative Example 2

The same procedure as in Example 1 was carried out except that thereaction time was shortened to one hour. The best part of the enzymereaction product gelled, and it was impossible to performcentrifugation. The liquid phase fraction was centrifuged to obtain 12 gof sediment, but the oil and fat content in the sediment was only 8.5%and it was impossible to recover a sufficient amount of oil.Electrophoretic analysis of the enzyme reaction solution showed that 46%of the original proteins had a molecular weight of approximately 120,000and 31% had a molecular weight of not less than 130,000.

EXAMPLE 2

Frozen krills were crushed by a food processor (mfd. by Nakabe Corp.)and 1,000 g thereof was weighed into a 2,000 ml beaker. The beaker wasfixed in a water bath and a variable-speed stirrer (EYELA Inc.) providedwith a stirring rod was set in the center of the beaker.

The crushed mass of krills was heated to 50° C. with stirring at 500rpm. When the mass being stirred reached 500° C., there were addedthereto 0.5% of an endo-type protease YP-SS (produced by Yakult ChemicalCo., Ltd.) and 0.5% of an exo-type Alloase P-10 (produced by YakultChemical Co., Ltd.), each being dissolved in a small quantity of water(% being based on the material to be treated). Vitamin E was also addedin an amount of 0.3% based on the oils and fats contained in thematerial. Then the mass was allowed to undergo enzyme reaction for onehour while maintaining the temperature at 50° C., followed by additional15-minute treatment at 90° C. to deactivate the enzymes. After thereaction, the resulting solution (O/W type emulsified composition) wasdiluted and subjected to electrophoretic analysis, which showedapproximately 65% of the original proteins came to have a molecularweight of 37,000.

The thus obtained emulsified composition was cooled, then put into atube of a high-speed centrifuge (mfd. by Hitachi Ltd.) and centrifugedat 10,000 rpm for 20 minutes.

After centrifuging, the centrifuge tube was taken out, the water-solublefraction in the upper part of the tube was transferred to a beaker, andthe solid composition was scraped out and transferred to a stainlesssteel-made plate vat. To 92 g of the obtained sediment, water was addedin an amount of 25% based on the solid composition to form an aqueoussolution, and the latter was dried by a laboratory spray dryer (mfd. byFreund Inc.) to give 33 g of dry powder.

As a result of the above operations, the following data were obtainedfrom the separated solids and water-soluble fraction.

Water content was determined by the Karl-Fischer method and oil and fatcontent was determined by the conventional gravimetric method involvingchloroform extraction. The data obtained from similar determinations ofraw krills and dry powder thereof are also shown.

Water Oil Raw krills 78.5% 3.1% Dry powder of krills 1.0 13.9 Dry powderof the solid composition 1.2 48.5 of the present invention Water-solublefraction 94.2 0.2 or less

As is seen from the above table, the oil and fat content in the drypowder of the solid composition of the present invention is higher by15.6 times and 3.5 times, respectively, than those in the raw krills andin the powder obtained by simply directly drying the krills. It will bealso seen that oils and fats are scantly in the water-soluble fraction.

EXAMPLE 3

The frozen tuna eyeballs were crushed by a food processor (mfd. byNakabe Corp.), and 1,000 g thereof was weighed into a 3,000 ml beakertogether with 1,000 g of water. The beaker was fixed in a water bath,and a variable-speed stirrer (mfd. by EYELA Inc.) provided with astirring rod was set in the center of the beaker.

The crushed mass of tuna eyeballs was heated to 50° C. with stirring at500 rpm. When the mass being stirred reached 50° C., there were addedthereto 0.5% of an endo-type protease YP-SS (produced by Yakult ChemicalCo., Ltd.) and 0.5% of an exo-type Alloase P-10 (produced by YakultChemical Co., Ltd.), each being dissolved in a small quantity of water(% being based on the material to be treated). Vitamin E was also addedin an amount of 0.3% based on the oils and fats contained in thematerial. Then the mass was allowed to undergo an enzyme reaction forone hour while maintaining the temperature at 50° C., followed byadditional 15-minute treatment at 90° C. to deactivate the enzymes. Theresulting reaction solution (O/W type emulsified composition) wasdiluted and then subjected to electrophoretic analysis, which showedapproximately 82.5% of the original proteins had a molecular weight of41,000 after the treatment.

The thus obtained emulsified composition was cooled, put into a tube ofa high-speed centrifuge (mfd. by Hitachi Ltd.) and centrifuged at 10,000rpm for 20 minutes.

On completion of centrifuging, the centrifuge tube was taken out, thewater-soluble fraction in the upper part of the tube was transferred toa beaker, and the solid composition was scraped out and transferred to astainless steel plate vat. To 620 g of the obtained solid composition,water was added in an amount of 25% based on the solid composition toform an aqueous solution, and the latter was dried by a laboratory spraydryer (mfd. by Freund Inc.) to give 226 g of dry powder. As a result ofthe above operations, the following data were obtained from theseparated solids and water-soluble fraction.

Water content was determined by the Karl-Fischer method and oil and fatcontent was determined by the conventional gravimetric method involvingchloroform extraction. The data obtained from similar determinations ofthe raw tuna eyes and dry powder thereof are also shown.

Water Oil Raw tuna eyeballs 66.5% 21.1% Dry powder of tuna eyeballs 1.634.6 Dry powder of solid composition of 1.2 55.7 the present inventionWater-soluble fraction (concentrated) 91.5 1.2

As is seen from the above table, the oil and fat content in the drypowder of the sediment according to the present invention is higher by2.6 times and 1.6 times, respectively, than those in the raw tunaeyeballs and the powder obtained by directly drying the tune eyeballs.It will be also seen that oils and fats are scantly in the water-solublefraction.

EXAMPLE 4

The frozen guts of sardine were crushed by a food processor (mfd. byNakabe Corp.) and 1,000 g thereof was weighed into a 3000 ml beaker, towhich 1,000 g of water was further added. The beaker was fixed in awater bath and a variable-speed stirrer (mfd. by EYELA Ltd.) providedwith a stirring rod was set in the center of the beaker.

The crushed mass of sardine guts was heated to 50° C. with stirring at500 rpm. When the mass reached 50° C., there were added thereto 0.3% ofendo-type Alkalase (produced by Novo Industry Ltd.), 0.3% of exo-typeAmano M (produced by Amano Pharmaceutical Co., Ltd.), 0.3% of exo- andendo-type Amano A (produced by Amano Pharmaceutical Co., Ltd.) and 0.1%of deaminase (produced by Daiwa Chemical Co., Ltd.) for improving thetaste and smell of the water-soluble fraction expected to be yielded,i.e. the extract, each being dissolved in a small quantity of water (%being based on the material to be treated). Vitamin E was also added inan amount of 0.3% based on the oils and fats contained in the material.Then the mass was allowed to undergo an enzyme reaction for 2 hourswhile maintaining the temperature at 50° C., followed by 15-minuteenzyme deactivating treatment at 90° C. The resulting solution (O/W typeemulsified composition) was diluted and subjected to electrophoreticanalysis, which showed approximately 67% of the original proteins had amolecular weight of 31,000 after the treatment.

The thus obtained emulsified composition was cooled, then put into atube of a high-speed centrifuge (mfd. by Hitachi Ltd.) and centrifugedat 10,000 rpm for 20 minutes.

After centrifuging, the centrifuge tube was taken out and 1,570 g of thewater-soluble fraction in the upper part of the tube was transferred toa beaker while the solid composition was scraped out and trans-ferred toa stainless steel plate vat. To 520 g of the obtained solid composition,water was added in an amount of 25% based on the composition to form anaqueous solution, and the latter was dried by a laboratory spray dryer(mfd. by Freund Inc.) to give 250 g of dry powder. As a result of theabove operations, the following data were obtained from the separatedsolids and water-soluble fraction.

Water content was determined by the Karl-Fischer method while the oiland fat content was determined by the conventional gravimetrical methodinvolving chloroform extraction. The data obtained from similardeterminations of the raw sardine guts and dry powder of sardine gutsare also shown.

Water Oil Raw sardine guts 67.4% 18.9% Dry powder of sardine guts 1.345.2 Dry powder of solid composition of 1.5 74.1 the present inventionWater-soluble fraction 93.1 1.8

As is seen from the above table, the oil and fat content in the drypowder of the solid composition according to the present invention ishigher by 3.9 times and 1.6 times, respectively, than those in the rawsardine guts and the powder obtained by simply directly drying thesardine guts. It will be also seen that oils and fats are scantly in thewater-soluble fraction.

INDUSTRIAL APPLICABILITY

The present invention provides a process for obtaining the water-solubleand water-insoluble useful substances from the waste of fishes andshellfishes by forming a novel oil-in-water (O/W) type emulsifiedcomposition containing oils and fats and solid proteins from the waste,and separating this emulsified composition into solid and liquid toobtain a novel solid composition. Use of the solid composition of thepresent invention as the starting material enables economical andindustrial extraction of the useful substances contained in the waste offishes and shellfishes which, in the past, has required troublesomesteps and has not necessarily been high in yield. Also, the presentinvention helps to pave the way for economical utilization of the wasteand produces a large effect in reducing environmental pollution by wastedisposal which has become more and more serious in recent years.

What is claimed is:
 1. A process for producing water-insolublesubstances derived from fishes and shellfishes, which comprises thesteps of: (a) treating portions of fishes and shellfishes containingsaid substances with a proteolytic enzyme under stirring to obtain anoil-in-water (O/W) type emulsified composition comprising, (i)water-soluble components which comprise water-soluble amino-acids,oligoproteins having a molecular weight of not greater than 30,000,vitamins and water-soluble minerals, and (ii) water-insolublecomponents, as solid matter, comprising oils and fats containingwater-insoluble unsaturated fatty acids and proteins having a molecularweight of 20,000 to 100,000, wherein at least 50% of all the proteins insaid emulsified composition have a molecular weight of 20,000 to100,000; (b) separating said emulsified composition into solid andliquid phases to obtain a solid composition; and (c) extracting saidsolid composition with an organic solvent.
 2. An oil-in-water (O/W) typeemulsified composition obtained by treating portions of fishes andshellfishes containing said composition of substances with a proteolyticenzyme under stirring, which comprises (i) water-soluble componentscomprising water-soluble amino-acids, oligoproteins having a molecularweight of not greater than 30,000, vitamins and water-soluble minerals,and (ii) water-insoluble components, as solid matter, comprising oilsand fats containing water-insoluble highly unsaturated fatty acids andproteins having a molecular weight of 20,000 to 100,000, wherein atleast 50% of all the proteins in the emulsified composition have amolecular weight of 20,000 to 100,000.
 3. A solid composition comprisingproteins having a molecular weight of 20,000 to 100,000 and oils andfats, obtained by separating into solid and liquid phases anoil-in-water (O/W) type emulsified composition, which is obtained bytreating portions of fishes and shellfishes containing said compositionwith a proteolytic enzyme under stirring; wherein the oil-in-water (O/W)type emulsified composition comprises water-soluble amino acids,oligoproteins having a molecular weight of not greater than 30,000,water-soluble minerals, oils and fats containing water-insoluble highlyunsaturated fatty acids and proteins having a molecular weight of 20,000to 100,000 wherein at least 50% of all the proteins in the emulsifiedcomposition have a molecular weight of 20,000 to 100,000.
 4. A processaccording to claim 1, wherein at least two enzymes selected from thegroup consisting of (1) an endo-type proteolytic enzyme, (2) an exo-typeproteolytic enzyme and (3) an endo- and exo-type proteolytic enzyme areused in combination as the proteolytic enzymes.
 5. A process accordingto claim 1, wherein the molecular weight of the proteins after treatmentis 5 30,000 to 50,000.
 6. A solid composition according to claim 3,wherein 50% or more of the proteins after treatment have a molecularweight of 30,000 to 50,000.
 7. A process according to claim 1, whereinthe portions comprise cuttlefish skin.
 8. A process according to claim1, wherein the portions comprise eyeballs or spawn of salmon or tuna. 9.A process according to claim 1, wherein the substance is a phospholipidtype unsaturated fatty acid.
 10. A process according to claim 1, whereinthe substance is phospholipid type DHA.
 11. A process according to claim1, wherein the solid matter after separation from the liquid phase isdried and then extracted with an organic solvent.
 12. A processaccording to claim 1, wherein the organic solvent is selected from thegroup consisting of ethanol, hexane, acetone and a mixture thereof. 13.Water-insoluble substances derived from fishes and shellfishes, obtainedby extracting the solid composition set forth in claim 3 with an organicsolvent.
 14. An edible composition containing at least 35% based on adry cake of the edible composition of a phospholipid, an unsaturatedfatty acid in an amount of at least 40% based on the whole fatty acidsin the phospholipid, and proteolytic enzyme-treated matter of fishes andshellfishes.
 15. A solid composition according to claim 2, wherein 50%or more of all the proteins in the emulsified composition have amolecular weight of 30,000 to 100,000.
 16. A solid composition accordingto claim 3, wherein 50% or more of all the proteins in the emulsifiedcomposition have a molecular weight of 30,000 to 100,000.